Discovery of Epoxysuccinates as Potent Covalent Inhibitors of SARS-CoV-2 Papain-like Protease

Abstract The papain-like protease (PLpro) of SARS-CoV-2, essential for viral processing and immune evasion, is a promising therapeutic target for coronavirus infections. Incorporating strategies used to enhance activity of non-covalent inhibitors, notably engagement of the BL2-groove, remote from the active site Cys-111, covalent PLpro inhibitors were designed. Epoxysuccinamide inhibitors demonstrated subnanomolar potency and 107-fold reduction in viral titer in cell cultures. The flexible hydrazide linker required for reaction of Cys-111 with the epoxysuccinamide warhead was susceptible to hepatic carboxylesterase metabolism. Replacement of hydrazide with alternative linkers was unsuccessful. Administered i.v., an epoxysuccinamide was well tolerated in mice. Although metabolically limited for targeting SARS-CoV-2 PLpro, the warhead was validated as a potent tool in the armamentarium of antivirals to confront emerging viruses.